An essential step during the life cycle of all retroviruses, including human immunodeficiency virus (HIV), is integration of a double-stranded DNA copy of the viral genome into a chromosome of the host cell. Two factors are critical for the integration process: the viral protein, integrase, and sequences at the ends of the linear viral DNA. Integrase removes two nucleotides from the 3' ends of a linear viral DNA molecule, and subsequently mediates a coupled cleavage-ligation reaction during which a staggered cut is made in the target DNA, and the resulting 5' ends of the target DNA are covalently joined to the recessed 3' ends of the viral DNA. The timing of the joining of the viral 5' ends to target DNA is presently not known, and the protein factors involved remain to be characterized. During integration, many sites in the host chromosome can be used as targets, although a wide variation in integration efficiency is observed among the target sites. The mechanism that determines target site specificity is not well understood. Since integration is required for retroviral replication and there is no recognized counterpart to integrase in normal cellular function, integration is an appealing target for developing specific inhibitors against retroviruses. The broad, long-term objective of this proposal is to further the understanding of the mechanism of HIV integration. The specific aims are (A) to examine target site selection during retroviral DNA integration, and (B) to study the kinetics and mechanisms of joining of the viral 5' end to target DNA, a poorly characterized final step of integration. The experimental design and methods for achieving these goals arc. (1) to construct chimeric proteins between HIV-1 and FIV integrases for determining the protein domain involved in selecting DNA sites for integration, and to use a PCR-based selection and amplification protocol for determining the DNA sequence optimal for integrase recognition, (2) to explore site-directed integration by studying in vitro and in vivo the activities and integration patterns of fusion proteins consisting of integraSe and a sequence-specific DNA binding protein, and (3) to use a novel strategy to study the timing of viral 3'- and 5'-end joining, and to develop an in vitro system for characterizing the 5'-end joining step and the factors required. The study may reveal potential novel targets for inhibitors and provide useful assays for drug screening. Information obtained from studying integration site selection may lead to a new approach for inserting exogenous genes at specific sites.